Genes that Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts

Background: Aortic valve stenosis (AVS) is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain under explored. Methods: Hydrogel scaffolds were designed to recapitulate key aspects of the valve tissue microenvironment and serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to pro-fibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA-sequencing was used to define pathways involved in driving sex-dependent activation. Interventions using small molecule inhibitors and small interfering RNA (siRNA) transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. Results: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker, alpha-smooth muscle actin (α-SMA), compared to male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-SMA stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase (RhoA/ROCK) signaling as a potential driver of this sex-dependent myofibroblast activation. Further, we found that genes that escape X-chromosome inactivation, such as BMX and STS (encoding for Bmx non-receptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation via RhoA/ROCK signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation via RhoA/ROCK signaling. Conclusions: Together, in vivo and in vitro results confirm sex-dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent AVS progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of AVS and help guide sex-based precision therapies.

Comprehensive Management of Cardiovascular Risk Factors for Adults With Type 2 Diabetes: A Scientific Statement From the American Heart Association

Cardiovascular disease remains the leading cause of death in patients with diabetes. Cardiovascular disease in diabetes is multifactorial, and control of the cardiovascular risk factors leads to substantial reductions in cardiovascular events. The 2015 American Heart Association and American Diabetes Association scientific statement, “Update on Prevention of Cardiovascular Disease in Adults With Type 2 Diabetes Mellitus in Light of Recent Evidence,” highlighted the importance of modifying various risk factors responsible for cardiovascular disease in diabetes. At the time, there was limited evidence to suggest that glucose-lowering medications reduce the risk of cardiovascular events. At present, several large randomized controlled trials with newer antihyperglycemic agents have been completed, demonstrating cardiovascular safety and reduction in cardiovascular outcomes, including cardiovascular death, myocardial infarction, stroke, and heart failure. This AHA scientific statement update focuses on (1) the evidence and clinical utility of newer antihyperglycemic agents in improving glycemic control and reducing cardiovascular events in diabetes; (2) the impact of blood pressure control on cardiovascular events in diabetes; and (3) the role of newer lipid-lowering therapies in comprehensive cardiovascular risk management in adults with diabetes. This scientific statement addresses the continued importance of lifestyle interventions, pharmacological therapy, and surgical interventions to curb the epidemic of obesity and metabolic syndrome, important precursors of prediabetes, diabetes, and comorbid cardiovascular disease. Last, this scientific statement explores the critical importance of the social determinants of health and health equity in the continuum of care in diabetes and cardiovascular disease.

Common Ancestry-specific Ion Channel Variants Predispose to Drug-induced Arrhythmias

Background: Multiple reports associate the cardiac sodium channel gene ( SCN5A ) variants S1103Y and R1193Q with type 3 congenital long QT syndrome (LQTS) and drug-induced LQTS. These variants are, however, too common in ancestral populations to be highly arrhythmogenic at baseline: S1103Y allele frequency is 8.1% in Africans and R1193Q 6.1% in East Asians. R1193Q is known to increase late sodium current (I Na-L ) in cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) but the role of these variants in modulating repolarization remains poorly-understood. Methods: We determined the effect of S1103Y on QT intervals among Africans in a large electronic health record. Using iPSC-CMs carrying naturally occurring or genome-edited variants, we studied action potential durations (APDs) at baseline and after challenge with the repolarizing potassium current (I Kr ) blocker dofetilide, and I Na-L and I Kr at baseline. Results: In 1479 African subjects with no confounding medications or diagnoses of heart disease, QT in S1103Y carriers was no different from that in non-carriers. Similarly, baseline APD was no different in cells expressing the Y allele (SY, YY cells) compared to isogenic cells with the reference allele (SS cells). However, I Na-L was increased in SY and YY cells and the I Na-L blocker GS967 shortened APD in SY/YY but not SS cells (p<0.001). I Kr was increased almost 2-fold in SY/YY cells compared to SS cells (tail current: 0.66±0.1 vs 1.2±0.1 pA/pF, p<0.001). Dofetilide challenge prolonged APD at much lower concentrations in SY (4.1 nM [IQR 1.5-9.3], n=11) and YY (4.2 nM [1.7- 5.0], n=5) than in SS cells (249 nM [22.3-2905], n=14, p<0.001 and p<0.01, respectively) and elicited afterdepolarizations in 8/16 SY/YY cells but only in 1/14 SS cells. R1193Q cells similarly displayed no difference in baseline APD but increased I Kr and increased dofetilide sensitivity. Conclusions: These common ancestry-specific variants do not affect baseline repolarization, despite generating increased I Na-L . We propose that increased I Kr serves to maintain normal repolarization but increases the risk of manifest QT prolongation with I Kr block in variant carriers. Our findings further emphasize the need for inclusion of diverse populations in the study of adverse drug reactions.

ZEB2 Shapes the Epigenetic Landscape of Atherosclerosis

Background: Smooth muscle cells (SMC) transition into a number of different phenotypes during atherosclerosis, including those that resemble fibroblasts and chondrocytes, and make up the majority of cells in the atherosclerotic plaque. To better understand the epigenetic and transcriptional mechanisms that mediate these cell state changes, and how they relate to risk for coronary artery disease (CAD), we have investigated the causality and function of transcription factors (TFs) at genome wide associated loci. Methods: We employed CRISPR-Cas 9 genome and epigenome editing to identify the causal gene and cell(s) for a complex CAD GWAS signal at 2q22.3. Subsequently, single-cell epigenetic and transcriptomic profiling in murine models and human coronary artery smooth muscle cells were employed to understand the cellular and molecular mechanism by which this CAD risk gene exerts its function. Results: CRISPR-Cas 9 genome and epigenome editing showed that the complex CAD genetic signals within a genomic region at 2q22.3 lie within smooth muscle long-distance enhancers for ZEB2 , a TF extensively studied in the context of epithelial mesenchymal transition (EMT) in development and cancer. ZEB2 regulates SMC phenotypic transition through chromatin remodeling that obviates accessibility and disrupts both Notch and TGFβ signaling, thus altering the epigenetic trajectory of SMC transitions. SMC specific loss of ZEB2 resulted in an inability of transitioning SMCs to turn off contractile programing and take on a fibroblast-like phenotype, but accelerated the formation of chondromyocytes, mirroring features of high-risk atherosclerotic plaques in human coronary arteries. Conclusions: These studies identify ZEB2 as a new CAD GWAS gene that affects features of plaque vulnerability through direct effects on the epigenome, providing a new thereapeutic approach to target vascular disease.

Direct Oral Anticoagulants versus Warfarin in Patients with Atrial Fibrillation: Patient-Level Network Meta-Analyses of Randomized Clinical Trials with Interaction Testing by Age and Sex

Background: Direct oral anticoagulants (DOACs) are preferred over warfarin for stroke prevention in atrial fibrillation (AF). Meta-analyses using individual patient data offer significant advantages over study-level data. Methods: We used individual patient data from the COMBINE AF database, which includes all patients randomized in the 4 pivotal trials of DOACs vs warfarin in AF (RE-LY, ROCKET AF, ARISTOTLE, ENGAGE AF-TIMI 48), to perform network meta-analyses using a stratified Cox model with random effects comparing standard-dose DOAC, lower-dose DOAC, and warfarin. Hazard ratios (95% CIs) were calculated for efficacy and safety outcomes. Covariate-by-treatment interaction was estimated for categorical covariates and for age as a continuous covariate, stratified by sex. Results: A total of 71,683 patients were included (29,362 on standard-dose DOAC, 13,049 on lower-dose DOAC, 29,272 on warfarin). Compared with warfarin, standard-dose DOACs were associated with a significantly lower hazard of stroke/systemic embolism (883/29312 [3.01%] vs 1080/29229 [3.69%]; HR 0.81, 95% CI 0.74-0.89), death (2276/29312 [7.76%] vs 2460/29229 [8.42%]; HR 0.92, 95% CI 0.87-0.97) and intracranial bleeding (184/29270 [0.63%] vs 409/29187 [1.40%]; HR 0.45, 95% CI 0.37-0.56), but no statistically different hazard of major bleeding (1479/29270 [5.05%] vs 1733/29187 [5.94%]; HR 0.86, 95% CI 0.74-1.01), whereas lower-dose DOACs were associated with no statistically different hazard of stroke/systemic embolism (531/13049 [3.96%] vs 1080/29229 [3.69%]; HR 1.06, 95% CI 0.95-1.19) but a lower hazard of intracranial bleeding (55/12985 [0.42%] vs 409/29187 [1.40%]; HR 0.28, 95% CI 0.21-0.37), death (1082/13049 [8.29%] vs 2460/29229 [8.42%]; HR 0.90, 95% CI 0.83-0.97), and major bleeding (564/12985 [4.34%] vs 1733/29187 [5.94%]; HR 0.63, 95% CI 0.45-0.88). Treatment effects for standard- and lower-dose DOACs versus warfarin were consistent across age and sex for stroke/systemic embolism and death, whereas standard-dose DOACs were favored in patients with no history of vitamin K antagonist use (p=0.01) and lower creatinine clearance (p=0.09). For major bleeding, standard-dose DOACs were favored in patients with lower body weight (p=0.02). In the continuous covariate analysis, younger patients derived greater benefits from standard-dose (interaction p=0.02) and lower-dose DOACs (interaction p=0.01) versus warfarin. Conclusions: Compared with warfarin, DOACs have more favorable efficacy and safety profiles among patients with AF.